Endotelial activation and flow-mediated vasodilation in young patients with breast cancer.

Endothelial activation and dysfunction may play a significant role in the progression of breast cancer. In our study we examined markers of endothelial activation (soluble ICAM-1, P-selectin, E-selectin) in 98 young patients with breast cancer (< 40 years). In 50 of them (and 20 age-matched controls) we also measured flow mediated vasodilation. Patients with breast cancer had significantly higher serum levels of soluble E-selectin, P-selectin and ICAM-1, P-selectin was higher in patients with larger tumors, node involvement and seemed to be apredictor of poor outcome. We were unable to find significant difference in the parameters of flow mediated vasodilation between patients with breast cancer and healthy subjects, although both peak blood flow (PBF) and flow mediated vasodilation (FMD) seemed to be skewed compared to healthy subjects toward mean and lower levels. Cluster analysis enabled us to distinguish several larger groups of patients with different degree of endothelial activation and function and different outcome. Group of patients with high E-selectin, high ICAM-1 (higher endothelial activation) and low VEGF (putative endothelial damage) had more frequently negative estrogen receptors and had worse outcome compared to the group of patients with lower E-selectin, lower ICAM-1 and mostly positive estrogen receptors. Further studies of larger groups of patients should help to identify the panel of endothelial markers which could help in predicting the outcome of young patients with breast cancer.

Menopause: clustering of metabolic syndrome components and population changes in insulin resistance.

AIM: The incidence of the metabolic syndrome (MS) in women rises rapidly during the menopause, substantially increasing their cardiovascular risk and mortality. The aim of the study was to analyze menopausal changes in individual MS components and the parameter of insulin resistance (HOMA-IR). METHODS: A random population sample of 909 women aged 45-54 years, resident in Prague 4, was examined in an epidemiological study. After excluding women with gynecological hormone therapy or surgical therapy, the two groups of women were compared: women of reproductive age (REPRO, n = 245) vs. naturally postmenopausal women (POSTm, n = 149). RESULTS: The incidence of MS rose significantly in menopause (REPRO/POSTm 22.9 ± 2.6%/38.3 ± 4.0%; p < 0.001). However, a detailed analysis among the five components defining MS showed that increases were only seen in waist circumference (p < 0.0001) and triglycerides (p < 0.001). There was no increase in the other components or HOMA-IR. A detailed analysis showed an increase in HOMA-IR at levels above the median (REPRO/POSTm: low HOMA-IR 0.9/0.9, not significant; high HOMA-IR 1.8/2.1, p < 0.001) and an increase in the incidence of MS just in these high levels of HOMA-IR and those rising during menopause (REPRO/POSTm: low HOMA-IR 13.8%/18.7%, not significant; high HOMA-IR 30.9%/57.3%, p < 0.0001). In menopause, there was an increase in the clustered incidence (accompanying MS) of each of the five MS components at the expense of isolated incidence (not accompanying MS). CONCLUSION: The acceleration of MS incidence at the onset of menopause may be accompanied by an increase in insulin resistance only in the population at highest risk. Reproductive women entering the menopause with an isolated MS component are at high risk for developing additional risk factors during menopause.

The OSBP-related proteins (ORPs): global sterol sensors for co-ordination of cellular lipid metabolism, membrane trafficking and signalling processes?

Protein families related to OSBP (oxysterol-binding protein) are present in eukaryotes from yeast to human. The functions of the ORPs (OSBP-related proteins) have remained largely enigmatic. Even though they have been implicated in the function of ERJs (endoplasmic reticulum junctions), it is evident that any single model for their mechanism of action is insufficient. The existing evidence points in many different directions, such as integration of sterol and sphingomyelin metabolism, regulation of neutral lipid metabolism, control of signalling cascades, regulation of secretory vesicle generation, and function in the microtubule-based motility of endo/lysosomes. Some of these functions could involve ERJ and non-vesicular transport of lipids, but this is unlikely to be the unifying feature. We believe, rather, that the common denominator for ORP function is acting as sterol sensors that relay information to a spectrum of cellular processes.

Determination of zinc in plant samples by isotope dilution inductively coupled plasma mass spectrometry.

Determination of zinc involved spiking with (68)Zn enriched solution, digestion by HNO(3)+H(2)O(2) in microwave decomposition unit, off-line separation of zinc on Chelex-100 column and measurement of ((64)Zn+(66)Zn)/(68)Zn isotope ratio on ICP-MS spectrometer with a quadrupole mass filter. After optimization of standard operation procedure (details are given) the method was validated. LOD was found to be 0.3 mug g(-1) for the procedure without zinc separation and 3.6 mug g(-1) for the procedure involving zinc separation, respectively. The accuracy of results was proved by analyses of several CRM and a primary solution of zinc, the concentration of which was verified by gravimetry and complexometric titration. Barium is the only element causing serious interferences and it must be removed from samples. The uncertainty budget is given together with the scheme of combined uncertainty calculation. The main uncertainty components are contamination during zinc separation and uncertainty of isotopic composition of natural zinc.

Custom-made interface between electrothermal vaporiser and inductively coupled plasma-mass spectrometer: determination of nickel by isotope dilution in plant samples.

ICP-mass spectrometer Perkin Elmer Elan 6000 was adapted for ETV analysis by coupling with GBC GF 3000 graphite furnace atomiser. Both instruments were connected by a 25 cm long PTFE tubing of 6 mm i.d. and a custom-made interface. The apparatus was applied to nickel determination using isotope dilution technique. The efficiency of analyte transfer through the interface was approximately 20%, however the incomplete transfer did not influence the blank values. Measured isotope ratio was very stable, whereas the magnitude of ion signal showed a little shift. CRM samples were analysed after microwave decomposition and by slurry technique as well. Obtained results were compared with those of pneumatic sample nebulisation after nickel separation on Chelex column. All tested techniques gave accurate results with comparable precision. However, the slurry technique allowed significant decreasing of the blank values and the limit of detection. The duration of analysis run can also be substantially shortened from several hours to a few minutes.

Monoclonal antibody to polycyclic aromatic hydrocarbons based on a new benzo[a]pyrene immunogen.

Benzo[a]pyrenebutyric acid (B[a]PBA) has been synthesized and covalently coupled to bovine serum albumin to generate monoclonal antibodies (Mab). A competitive indirect enzyme-linked immunosorbent assay (ELISA) for polycyclic aromatic hydrocarbons (PAH) has been developed with Mab B[a]P-13. It was shown by testing with 21 parent PAH and 10 compounds carrying methyl, hydroxy, or butyric acid functions that the antibody had broad specificity. Highest affinity was found for four- to six-ring PAH. Different organic co-solvents were tested. No loss in sensitivity, compared with controls in PBS, were found with methanol, dimethyl sulfoxide, and glycerol at final concentrations of 5 to 10%. Further, an observation was made that a modification (fine-tuning) of the affinity and specificity of the antibodies was possible by changing the type of the added organic co-solvent. The high susceptibility of the ELISA with regard to inorganic ions might be an indication of a more hydrophilic binding pocket e.g. involving a pi-cation interaction. Investigation of the effect of pH revealed that for pH between 6 to 9 there was no noticeable impairment. With an LOD as low as 30 pg per well for B[a]P the sensitivity of the ELISA is sufficient for analyses of solvent extracts of many environmental samples. As an example, the determination of a PAH sum parameter, given as B[a]P-equivalents, in crude aerosol extracts by both ELISA and HPLC revealed good correlation (r2=0.717) but approximately five-fold overestimation by the immunochemical method, obviously as a result of cross-reacting analytes.

Determination of copper, selenium and zinc in human blood by inductively coupled mass spectrometry: the sources of uncertainty and variability of results.

The concentration of Cu, Se and Zn in whole human blood and plasma was analysed by inductively coupled plasma mass spectrometry. Method involved dilution of sample by diluted nitric acid and Triton X-100 and measurement of 65Cu, 77Se and 66Zn intensities. Calibration solutions were spiked by main interferents. In order to evaluate suitability of the method for recognition of alternation of trace elements concentration, the uncertainty of results was estimated. The major part of uncertainty was due to repeatability, the other source (calibration and interferences) were found to be of lesser importance. The resulting uncertainty of concentration of mentioned trace elements is low as compared to between individual variability. Short-term (within day) and long-term (between day) variability of trace elements concentration was investigated and compared with between individual variability too. The time variability was negligible except for Zn concentration. The alternations of trace elements concentration in patients with several renal diseases were investigated. Only in case of patients in end-stage renal disease: hemodialyzed patients and patient treated by continuous ambulatory peritoneal dialysis the significant alternations were proved.

[Linear discrimination analysis in blood pressure monitoring]. / Vyuzití lineární diskriminacní analýzy pri monitorování krevního tlaku.

The 24-hours blood pressure monitoring (BPM) is a reliable technique largely used for hypertension diagnosis in clinical practice. Presence of the lack of a nocturnal decrease in blood pressure values is one differentiating criterion between essential and secondary hypertension. New useful method of mathematical and statistical processing of BPM data using the linear discriminant analysis (LDA) was described. This statistical analysis was applied for 123 BPM measurements from patients with essential hypertension without and with different kidney diseases including hemodialyzed patients. The control group was normotensive healthy volunteers. The LDA method successfully separate group of patients with essential hypertension from patients with secondary renal hypertension but the error of return ranking to these groups was 28.5%. The recalculation for groups-controls, hypertensive patients and hypertensive hemodialyzed patients leads to decreasing of the error of return ranking to 5.7%. This retrospective study of nonhomogeneous groups could not clearly differentiate groups of hypertensive patients. The new prospective study on accurately definite groups of patients can eliminate these problems.

Trace elements in hemodialysis and continuous ambulatory peritoneal dialysis patients.

Alterations in blood and tissue concentrations of trace elements in patients with chronic renal failure have been extensively investigated. Selenium, zinc and copper are elements which play an important role in biological systems as components of proteins, enzymes and antioxidants. The concentrations of selenium, zinc and copper were determined in the plasma, erythrocytes and whole blood of patients on regular hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) treatment using the method of inductively coupled plasma mass spectrometry (ICP-MS). Analysis of isotopes 77Se, 66Zn and 65Cu was performed. Methodology presents the major limitation to valid studies on trace element levels in biological materials. One of the widely used contemporary techniques is ICP-MS. It is the most sensitive one and has a high dynamic range. The selenium concentration in the studied compartments (plasma 46.1 +/- 3.0 vs. 78.0 +/- 3.4 microgram/l, p < 0.001; erythrocytes 90.4 +/- 6.5 vs. 134.2 +/- 7.6 microgram/l, p < 0.01; whole blood 67.3 +/- 3.1 vs. 106.4 +/- 3.4 microgram/l, p < 0.001) was significantly lower in HD patients compared to healthy controls. The same result was observed in plasma (63.2 +/- 5.8 vs. 78.0 +/- 3.4 microgram/l, p < 0.05) and whole blood (82.7 +/- 7.4 vs. 106.4 +/- 3.4 microgram/l, p < 0.01) from CAPD patients, but the selenium level of erythrocytes in CAPD patients was the same as in the control group (126.0 +/- 8.8 vs. 134. 2 +/- 7.6 microgram/l). The cooper content of erythrocytes was lower in HD patients than in controls (0.55 +/- 0.02 vs. 0.66 +/- 0.01 mg/l, p < 0.01) and CAPD groups (0.55 +/- 0.02 vs. 0.68 +/- 0.02 mg/l, p < 0.001). There were no differences in copper content in plasma (HD 1. 02 +/- 0.06; CAPD 1.11 +/- 0.09; controls 1.02 +/- 0.05 mg/l) and whole blood (HD 0.87 +/- 0.04; CAPD 0.90 +/- 0.05; controls 0.85 +/- 0.02 mg/l) in HD and CAPD patients and healthy controls. The zinc concentration was increased in the whole blood of CAPD patients (6. 68 +/- 0.36 vs. 5.52 +/- 0.11 mg/l, p < 0.001) and erythrocytes of HD (12.30 +/- 0.23 vs. 10.11 +/- 0.42 mg/l, p < 0.001), and CAPD groups (13.71 +/- 0.56 vs. 10.11 +/- 0.42 mg/l, p < 0.001) compared to controls. However, the plasma zinc concentration was lower in HD patients compared to blood donors (0.69 +/- 0.03 vs. 0.92 +/- 0.03 mg/l, p < 0.001) and CAPD patients (0.69 +/- 0.03 vs. 0.95 +/- 0.04 mg/l, p < 0.001). We did not find a significant increase in trace elements in whole blood after HD. These results suggest differences between plasma, erythrocytes and whole blood concentrations of the studied trace elements. The levels of trace elements are altered by HD and CAPD. A modern precise method with high accuracy, ICP-MS, which was used in our study, eliminated analytical errors and possible interferences.

Constitutive intra-articular expression of human IL-1 beta following gene transfer to rabbit synovium produces all major pathologies of human rheumatoid arthritis.

To investigate the pathophysiologic effects of chronically elevated intra-articular levels of IL-1 beta, we used an ex vivo gene transfer method to deliver and express human IL-1 beta (hIL-1 beta) in the knee joints of rabbits. Expression of hIL-1 beta resulted in a severe, highly aggressive form of arthritis analogous to chronic rheumatoid arthritis in humans. Intra-articular manifestations included intense inflammation, leukocytosis, synovial hypertrophy and hyperplasia, and highly aggressive pannus formation with erosion of the articular cartilage and periarticular bone. Systemic effects were also observed, including diarrhea, fever, weight loss, and an increased erythrocyte sedimentation rate. In addition, the hIL-1 beta was found to induce elevated levels of both rabbit IL-1 beta and TNF-alpha in synovial fluid. Following the loss of hIL-1 beta transgene expression between 14 and 28 days post-transplantation, many of these changes began to normalize. These results suggest that chronically elevated intra-articular levels of IL-1 beta alone are sufficient to produce virtually all the pathologies found in rheumatoid arthritis, and furthermore, demonstrate that gene transfer can be used to investigate the roles of specific gene products in the pathogenesis of arthritis.

Quantitation of collagen types I, III and V in tissue slices by capillary electrophoresis after cyanogen bromide solubilization.

A method for the determination of the proportions of major fiber-forming collagens (types I, III and V) in soft connective tissue was elaborated. The method is based on the release of insoluble collagen by CNBr with subsequent separation of the arising peptides. For routine application the peptides are separated by capillary electrophoresis (50 mM phosphate pH 2.5, 15 kV, 50 degrees C, 70/60 cm x 70 microns I.D. capillary with UV detection at 200 nm). Quantitation of collagen type I can be done either on the basis of spiking the sample with a peptide mixture obtained from a known amount of collagen type I, or by spiking the sample with an equimolar mixture of the two peptides [alpha 1(I)CB2 and alpha 1(I)CB4] (constituting a fused peak) along with alpha 1(III)CB2 and alpha 1(V)CB1. Compared to the previously published methods the procedure is faster and does not require isolation of marker peptides by tedious chromatographic procedures in a preceding preparatory step. Good results are obtained within a wide range of run buffer concentrations and applied voltages; conversely, intensive cleaning of the capillary after every three runs is recommended with a new capillary after 20-30 runs.

[Use of multivariate analysis in medical chemistry]. / Vyuzití vícerozmerné analýzy rozptylu v lékarské chemii.

Alternations in trace elements concentrations are intensively studied because of their possible role in pathogenesis and progress of diseases. Three groups of patients were investigated: haemodialysis patients (HD) (n = 17), continual ambulatory peritoneal dialysis patients (CAPD) (n = 11), and control group of blood donors (n = 12). They were analyzed for Se, Zn and Cu concentration in plasma and erythrocytes by inductively--coupled plasma mass spectroscopy. Distribution of analyzed elements between these blood compartments was found different in all groups of studied patients. Erythrocytes were enriched by Se (twofold higher concentration compared to plasma) and by Zn (10-20 fold higher concentrations compared to plasma). On the other hand, human plasma was enriched by Cu (approximately twofold higher concentration then in erythrocytes). Results of analyses were processed by multivariate analysis of variance (MANOVA). When only results of plasma analysis were involved into MANOVA, differences between HD patients and other two groups were found whereas CAPD and control group were not distinguished each other. However, these two groups were mutually differenced when MANOVA comprised all trace elements concentrations: both plasma and erythrocytes. Methods of multivariate statistic are able to study not only the individual variables but even their mutual relations and their medical applications are very useful.

Separation of common nucleotides, mono-, di- and triphosphates, by capillary electrophoresis.

A capillary electrophoretic procedure for the separation of eleven nucleotides, 5'-mono-, di- and triphosphates of adenosine, guanosine, cytidine and uridine, has been developed. All eleven analytes can be separated in a fused-silica capillary (63 cm to the detector, I.D. 75 microns) at 20 kV in a 0.02 mol 1(-1) phosphate-borate buffer (pH 8.0-9.0) with a separation factor > or = 1. The values of the Offord parameter calculated for individual nucleotides predict that monophosphates will migrate faster than triphosphates, and in turn triphosphates will precede diphosphates. By analogy, faster electroosmotic mobility (lower electromigration) of purine nucleotides (AP, GP) can be explained by a more voluminous structure of purine derivatives (two aromatic rings as compared to pyrimidines). Generally speaking, all compounds separated follow the Offord equation assuming that the triphosphate derivatives are ionized to the third degree forming HL(3-) anions. This assumption is in agreement with the current knowledge about protolytic equilibria of polyphosphates. The only exception to this rule is faster migration of guanosine-5'-triphosphate (GTP) preceding uridine-5'-monophosphate (UMP) which is ascribed in part to the larger molecule of GTP and the two additional OH-groups bound to the pyrimidine ring of UMP.

Qualitative analysis of green coffee by infrared spectrometry.

Principal component analysis and linear discriminant analysis (LDA) were applied to the mid-infrared spectra for the qualitative analysis of the variety of green coffee (Arabica and Robusta). It is shown that the KBr pellet technique in combination with the LDA method can successfully be used for the identification of sample origin.

Cloning, expression, and functional characterization of rat MIP-2: a neutrophil chemoattractant and epithelial cell mitogen.

Macrophage inflammatory protein-2 (MIP-2) is a member of a family of cytokines that play roles in inflammatory, immune, and wound healing responses. To clone the cDNA for rat MIP-2, RNA was isolated from the lungs of Fischer 344 rats after instillation of lipopolysaccharide. Reverse transcription-polymerase chain reaction was performed by using synthetic oligonucleotide primers designed from the mouse MIP-2 cDNA sequence. A cDNA containing the coding region of rat MIP-2 was cloned and sequenced. Comparison to the mouse MIP-2 cDNA demonstrated 90.3% homology at the nucleotide level and 86% homology at the amino acid level. The rat MIP-2 cDNA was expressed in Escherichia coli and protein evaluated for bioactivity. The recombinant rat MIP-2 was chemotactic for rat neutrophils but did not stimulate migration of rat alveolar macrophages or human peripheral blood eosinophils or lymphocytes. In addition, the recombinant rat MIP-2 and the related rat chemokine, KC/CINC stimulated proliferation of rat alveolar epithelial cells but not fibroblasts in vitro.

Suppression of intra-articular responses to interleukin-1 by transfer of the interleukin-1 receptor antagonist gene to synovium.

We have developed an ex vivo method for delivering genes to the synovial lining of joints and expressing them intra-articularly. The present studies were designed to determine whether transfer of a human interleukin-1 receptor antagonist protein (IRAP) gene by this method was able to antagonize the intra-articular actions of interleukin-1. Intra-articular injections of human recombinant interleukin-1 beta (hrIL-1 beta) into the knees of control rabbits provoked a marked leukocytic infiltrate into the joint space, severe synovial thickening and hypercellularity, and loss of proteoglycans from articular cartilage. Genetically modified knees contained several nanograms of human IRAP and inhibited each of these effects of IL-1 beta. These data demonstrate for the first time that delivery of an appropriate gene to joints can prevent intra-articular pathology. Such findings permit cautious optimism about the eventual development of a gene treatment for arthritis and other disorders of the joint.

Intraarticular expression of biologically active interleukin 1-receptor-antagonist protein by ex vivo gene transfer.

Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-1ra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular injection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for approximately 5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1 beta. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by gene-transfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy.

[Ablation of the accessory bundle of Kent using radiofrequency current in a 17-year-old boy with type A Wolff-Parkinson-White syndrome]. / Prerusení akcesorního Kentova svazku radiofrekvencním proudem u sedmnáctileteho chlapce s WPW syndromem typu A.

The authors describe the in Czechoslovakia so far not used therapeutic catheterization ablation method of the accessory atrioventricular pathway in patients with tachycardia with the Wolff-Parkinson-White syndrome. They performed successful ablation of the accessory bundle of Kent located by electrophysiological examination behind the left atrium and left ventricle in a 17-year-old boy with WPW Syndrome type A. They severed the accessory pathway by radiofrequency current using a catheter inserted from a retrograde approach into the left ventricle beneath the mitral valve. After ablation the boy suffers no longer from tachycardia, the ECG tracing in devoid of preventricular preexcitation, although he has no medicamentous treatment.